1
Supplementary Methods
2
Details of surgical procedure and sample collection
3
Children were randomised to receive adenoidectomy + myringotomy (n=5/11) or adenoidectomy +
4
tympanostomy tubes (n=6/11) pursuant to the surgical study. All clinical specimens were collected under
5
general anaesthesia. Intraoperative collection and processing of samples was supervised by the principle
6
author to ensure suitability for DNA based microbiological methods.
7
Inclusion criteria for the surgical study were:
8
1.
Indigenous children aged 3 - 10 years old living in remote communities, and
9
2.
OME/AOM that has been present for ≥6 months and failed medical treatment.
10
The clinical criteria for the diagnosis of OME was the presence of an immobile tympanic membrane on
11
pneumatic otoscopy, supported by an air-bone gap on audiometry (conductive hearing loss >30dB) and a Type
12
B tympanogram.
13
Wax and other debris was removed from the ear canal using standard techniques. Prior to myringotomy, no
14
sterilisation of the ear canal was undertaken (myringotomy: a small surgical incision in the tympanic
15
membrane). Following myringotomy, a suction catheter removed the MEF through the myringotomy. The
16
effusion was collected into an Argyle Specimen Trap (Covidien, Massachusetts USA) by aspiration of 2ml of
17
saline through the suction catheter. Nasopharyngeal swabs were collected according to the technique
18
recommended by World Health Organisation Pneumococcal Working Group [1]. A sterile Paediatric FLOQ
19
Swab (Copan, California USA) was passed along the floor of the nasal cavity into the nasopharynx of each
20
child, remaining in situ for 5 seconds while being rotated 180 degrees to saturate the swab. The swab was
21
removed and placed into a skim milk tryptone glucose glycerol broth (STGGB). Adenoidectomy was then
22
undertaken using a curette technique. All specimens were stored on ice until the end of the surgical procedure,
23
and then transferred to a -70 C freezer. 11 adenoid swabs were taken from the adenoid biopsies prior to DNA
24
extraction. In addition to the 22 MEFs, 11 NP swabs, 11 adenoid biopsies and 11 adenoid swabs (total 55
25
samples), a number of replicate MEF samples were collected for 16S rRNA sequencing. A total of 8 swabs of
26
suction tubing were included in the clinical samples. Additionally, for all MEF samples, 0.25ml of the 2ml
27
solution was transferred into an STGGB tube. 10/22 MEF samples in STGGB (bilateral samples from 5
28
children) were therefore included in the clinical samples. Accordingly, 73 clinical samples were sent for DNA
1
29
extraction. The 18 samples comprising of MEF replicates (8 MEF Tube and 10 STGGB samples) were only
30
used for the purpose of establishing correlation plots to identify contaminant OTUs and were not used for any
31
further downstream analysis.
32
33
DNA extraction from clinical samples
34
All samples were thawed on ice. After thawing, a swab of the adenoid tissue surface was prepared. The swab
35
was then placed into 1 mL of sterile STGGB media and vortexed at full speed for 1 minute. DNA was then
36
extracted from 200 µL of MEF, 400 µL of NP swab media, 1mL of adenoid swab media and 8-30 mg of
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adenoid tissue.
38
For MEF and swab media specimens, cellular material was pelleted by centrifugation at 7400 x g at 4ºC for 3
39
mins. Pellets were then resuspended in 600 μL lysis buffer consisting of Qiagen Buffer AL (Qiagen, Victoria,
40
Australia), 2.9 mg ml-1 lysozyme, 0.075 mg ml-1 mutanolysin and 0.019 mg ml-1 lysostaphin. The mixture was
41
then incubated at 37°C for 30 min.
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Adenoid tissue samples were homogenised using a micropestle after addition of 50 µL of lysis buffer.
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Following homogenisation, a further 550 µl of lysis buffer was added and the sample mixed by pipetting. The
44
mixture was then incubated at 37°C for 30 min.
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All samples were then mechanically lysed using a FastPrep instrument (MP Biomedicals, CA, USA) with
46
Lysing Matrix B (MP Biomedicals, CA, USA). Bead-beating was performed for 30 sec at 6.0 m sec-1. The
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supernatant was then taken and 45 µl proteinase K (20 mg ml-1) was added. The samples were then incubated
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at 65°C for 10 min. Subsequently, 200 μL of ethanol was added and the genomic DNA was purified using a
49
QIAamp DNA Mini Kit (Qiagen), as per the manufacturer’s tissue protocol (QIAamp Micro Handbook,
50
version 08/2003). DNA concentration and purity were measured using a NanoDrop spectrophotometer
51
(Thermo Fisher Scientific, Australia). Samples with DNA concentrations below the dynamic range of the
52
NanoDrop spectrophotometer were analysed using Qubit High-Sensitivity (HS) dsDNA Assay kit (Life
53
Technologies, Australia).
54
55
Real-time quantitative PCR
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Quantitative PCR to estimate the total bacterial load was performed using the primers of Nadkarni et al. [3]
2
57
which amplify a 466-bp region between positions 331-797 of the 16S rRNA gene, based on E. coli numbering.
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The assay was performed as described previously [2]. Each 10µL qPCR reaction included 1X SensiMixTM
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SYBR® reagent (Bioline), 300nM of each primer and 1µL of template DNA. The qPCR was performed using
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a Rotor-Gene 6000 real-time thermocycler. The reaction conditions were an initial hold at 50ºC for 2 min
61
followed by incubation at 95ºC for 10 min then 35 cycles of 95ºC for 15 s, 58ºC for 15 s and 72ºC for 45 s.
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Melt-curve analysis was then done between 80ºC-90ºC with 0.1ºC steps. As multiple amplicons form during
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universal 16S rRNA gene amplification, it was not possible to define a single melt-curve dissociation
64
temperature that could be used to differentiate specific from non-specific amplicons. To overcome this
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limitation, the entire melt-curve was considered. Replicate analyses with irreproducible melt-curves were
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considered indicative of non-specific amplification. Genomic DNA from the S. pneumoniae ATCC49619
67
reference isolate was used to prepare the standard curve (1:10 serial dilution from 2000ng-200fg). Thus, the
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assay estimated total bacterial load assuming four ribosomal operons per cell [3]. The total bacterial load
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qPCR limit of detection was 90 cells based on an S. pneumoniae genome size of ~2Mb [GenBank:AE005672].
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qPCR raw data and standard curves were prepared using the Rotor-Gene 6000 software (Corbett Research;
71
version 1.7).
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73
16S rRNA amplicon library construction and sequencing
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Barcoded amplicon libraries for the bacterial community analysis on the Illumina MiSeq platform were
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generated using degenerate primers targeting the V1 and V3 hypervariable region of the bacterial 16S rRNA
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gene and Nextera XT index kit (Illumina, Inc., Victoria, Australia).
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Amplicons
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TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGTTTGATCMTGGCTCAG-3') and 519R (5'-
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GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTNTTACNGCGGCKGCTG-3')
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overhang Illumina adapter consensus sequences as shown in underlines. Initially, PCR reactions were
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performed on a Veriti 96-well Thermal Cycler (Life Technologies, Australia) with 12.5 µl of 2X KAPA HiFi
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Hotstart Ready Mix (KAPA Biosystems, MA, USA), 12.5 ng of total DNA template, 0.5 µM of each primer
83
and nuclease-free water to a total volume of 25 µl. Samples with low DNA yield (less than 2 ng µl-1, as
84
measured from the Qubit HS dsDNA assay kit) were carried out in a 50 µl PCR reaction to achieve a starting
were
generated
using
fusion
degenerate
primers
27F
with
(5'-
ligated
3
85
DNA amount of 12.5 ng. The PCR reactions were performed in the following program; initiation enzyme
86
activation at 95°C for 3 min, followed by 25 cycles consisting of denaturation at 95°C for 30 sec, annealing at
87
55°C for 30 sec, and extension at 72°C for 30 sec. After 25 cycles, the reaction was completed with a final
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extension of 7 min at 72 °C. The 640 bp 16S amplicons were purified using 20 µl Agencourt Ampure XP
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magnetic beads (Beckman Coulter, Inc., New South Wales, Australia) according to the manufacturer's
90
instructions, with exceptions for the 50 µl PCR reaction, where 40 µl of the bead solution was added to the
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sample to maintain the final polyethylene glycol concentration. The 16S amplicons were eluted from the
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magnetic beads in 52.5 µl of 10 mM Tris-HCl, pH8.0.
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The Illumina Nextera XT Index kit with dual 8 bases indices were used to allow for multiplexing. Two unique
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indices located on either end of the amplicon were chosen based on the Nextera dual-indexing strategy. To
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incorporate the indices to the 16S amplicons, PCR reactions containing 25 µl of KAPA HiFi HotStart Ready
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Mix, 5 µl of each i5 and i7 index (Illumina), 5 µl of purified amplicons and nuclease-free water were mixed to
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a total volume of 50 µl, and were performed on a Veriti 96-well Thermal Cycler (Life Technologies). Cycling
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conditions consist of one cycle of 95°C for 3 min, followed by eight cycles of 95°C for 30 sec, 55°C for 30
99
sec, and 72°C for 30 sec, followed by a final extension cycle of 72°C for 5 min. The barcoded amplicons were
100
then purified using 56 µl of Agencourt Ampure XT magnetic beads (Beckman Coulter, Inc.) according to the
101
instructions of the manufacturer. The barcoded libraries were eluted from the magnetic beads in 27.5 µl of 10
102
mM Tris-HCl, pH8.0. Following library preparation, samples with low amplicon concentration (<2ng/µl) were
103
excluded from sequencing.
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Prior to library pooling, the barcoded libraries were quantified using Qubit HS dsDNA assay kit (Life
105
Technologies). Libraries were mixed in approximately equal concentrations to ensure an even representation
106
of reads per sample. The size of the pooled libraries were verified using Agilent DNA 1000 Analysis kit
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(Agilent Technologies, Inc., CA, USA) on the Agilent 2100 Bioanalyzer system (Agilent Technologies). The
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670 bps barcoded libraries were denatured at 4 nM before diluting to a final concentration of 8 pM. The
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libraries were then spiked with 20% PhiX control (Illumina), and were sequenced by 2 x 300 bp paired-end
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sequencing on the MiSeq platform using MiSeq v3 Reagent Kit (Illumina) at the Flinders Genomics Facility,
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Adelaide, Australia.
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4
113
114
References
1. Satzke C, Turner P, Virolainen-Julkunen A, Adrian PV, Antonio M, Hare KM, Henao-Restrepo AM,
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Leach AJ, Klugman KP, Porter BD, Sá-Leão R, Scott JA, Nohynek H, O'Brien KL; WHO
116
Pneumococcal Carriage Working Group: Standard method for detecting upper respiratory
117
carriage of Streptococcus pneumoniae: updated recommendations from the World Health
118
Organization Pneumococcal Carriage Working Group. Vaccine 2013, 32:165-79.
119
2. Marsh RL, Binks MJ, Beissbarth J, Christensen P, Morris PS, Leach AJ, Smith-Vaughan HC:
120
Quantitative PCR of ear discharge from indigenous Australian children with acute otitis media
121
with perforation supports a role for Alloiococcus otitidis as a secondary pathogen. BMC Ear,
122
Nose and Throat Disord 2012, 12:11.
123
3. Nadkarni MA, Martin FE, Jacques NA, Hunter N: Determination of bacterial load by real-time
124
PCR using a broad-range (universal) probe and primers set. Microbiol 2002, 148:257-266.
125
4. Smith-Vaughan H, Byun R, Nadkarni M, Jacques NA, Hunter N, Halpin S, Morris PS, Leach AJ:
126
Measuring nasal bacterial load and its association with otitis media. BMC Ear Nose and Throat
127
Disord 2006, 10:10.
128
5. Binks M, Cheng A, Smith-Vaughan H, Sloots T, Nissen M, Whiley D, McDonnell J, Leach A: Viral-
129
bacterial co-infection in Australian Indigenous children with acute otitis media. BMC Infect Dis
130
2011, 11:161.
131
5
132
133
Figure S1. Effect of subsampling on microbiota diversity. Box and whisker plot of Simpson’s Index of
134
Diversity (1-D) at four levels of subsampling. Diversity levels are shown for the 21 samples that had ≥ 2000
135
reads, post-filtering.
136
137
138
139
Figure S2. Host-read contamination. Bar plot of median relative abundance of host reads (Human Genome
140
GRcH38) in each sample type. Error bars represent interquartile range.
141
6
142
Table S1. Composition of multi-template control.
143
* Relative abundance as a percentage of 16S rRNA gene copy number.
144
Species
Strain ID
Alloiococcus otitidis
Bacteroides fragilis
Escherichia coli
Haemophilus influenzae
Haemophilus parainfluenzae
Klebsiella pneumoniae
Moraxella catarrhalis
Neisseria meningitidis
Proteus mirabilis
Pseudomonas aeruginosa
Staphylococcus aureus
Streptococcus mitis
Streptococcus pneumoniae
ATCC51267
ATCC23748
ATCC25922
ATCC49247
ATCC7901
MSHR110128
ATCC8176
ATCC13090
ATCC12453
ATCC27853
ATCC25923
ATCC49619
ATCC6249
Relative
abundance*
0.43
10.02
0.67
15.2
1.88
37.62
10.39
7.39
0.39
2.97
5.64
0.05
7.36
7
145
Table S2. Composition of multi-template control, used in the assessment of reference databases.
Expected
Family
Expected Genus
Expected
Enterobacteriaceae
44.36% Klebsiella
43.36%
Bacteroidaceae
13.63% Bacteroides
13.63%
Moraxellaceae
7.54% Moraxella
7.54%
Pseudomonadaceae
7.24% Pseudomonas
7.24%
Pasteurellaceae
8.22% Haemophilus
8.22%
Streptococcaceae
6.20% Streptococcus
6.20%
Staphylococcaceae
6.19% Staphylococcus
6.19%
Neisseriaceae
6.19% Neisseria
6.19%
Carnobacteriaceae
0.29% Escherichia
0.71%
Proteus
0.36%
Alloiococcus
0.29%
146
147
8
148
Table S3. Analysis of multi-template control sequence data through comparison with Greengenes reference database.
149
* = BLAST identity at >95%.
GREENGENES (OTUs with relative abundance >.40%)
#OTU ID
183603
Seq.
14426
27.22% Bacteroidaceae
Bacteroides
Is the highest classification
assigned by Greengenes correct
according to NCBI?
Yes
Rel.
Abund.
Family
Genus
N.R.OTU0
9093
17.16% Unassigned
Unassigned
not assigned (Moraxella)*
N.R.OTU1
8120
15.32% Pasteurellaceae
Haemophilus
Yes
197286
6936
13.09% Enterobacteriaceae
Unassigned
Yes
4365567
3254
6.14% Neisseriaceae
Neisseria
Yes
222184
2167
4.09% Pseudomonadaceae
Pseudomonas
Yes
4376233
667
1.26% Enterobacteriaceae
Unassigned
Yes
4305793
637
1.20% Pasteurellaceae
Haemophilus
Yes
4455250
482
0.91% Streptococcaceae
Streptococcus
Yes
N.C.R.OTU1333
418
0.79% Unassigned
Unassigned
not assigned (Haemophilus)*
560629
329
0.62% Enterobacteriaceae
Proteus
Yes
N.C.R.OTU356
289
0.55% Unassigned
Unassigned
not assigned (Moraxella)*
N.C.R.OTU1974
253
0.48% Unassigned
Unassigned
not assigned (Moraxella)*
N.C.R.OTU3450
235
0.44% Unassigned
Unassigned
not assigned (Neisseria)*
N.C.R.OTU6077
220
0.42% Unassigned
Unassigned
not assigned (Staphylococcus)*
N.C.R.OTU1205
219
0.41% Unassigned
Unassigned
not assigned (Moraxella)*
N.C.R.OTU231
212
0.40% Unassigned
Unassigned
not assigned (Moraxella)*
150
9
151
Table S4. Analysis of multi-template control sequence data through comparison with SILVA reference database.
152
* = NCBI BLAST identity at >95%.
SILVA (OTUs with relative abundance >.40%)
26.71% Bacteroidales (order)
Unassigned
Is the highest classification
assigned by SILVA correct
according to NCBI?
Yes
8993
16.65% Moraxellaceae
Moraxella
Yes
N.R.OTU0
8827
16.35% Pasteurellaceae
Haemophilus
Yes
EU775630
4783
8.86% Enterobacteriaceae
Unassigned
Yes
GP740200
3806
7.05% Neisseriaceae
Neisseria
Yes
GQ258634
2155
3.99% Pseudomonadaceae
Pseudomonas
Yes
EU775715
2059
3.81% Enterobacteriaceae
Unassigned
Yes
FJ558078
614
1.14% Streptococcaceae
Streptococcus
Yes
EU775611
576
1.07% Enterobacteriaceae
Unassigned
Yes
N.R.OTU11
399
0.74% Pasteurellaceae
Haemophilus
Yes
N.R.OTU10
352
0.65% Unassigned
Unassigned
not assigned (Moraxella)*
HQ407310
340
0.63% Enterobacteriaceae
Proteus
Yes
N.R.OTU16
321
0.59% Unassigned
Unassigned
not assigned (Moraxella)*
N.R.OTU8
262
0.49% Unassigned
Unassigned
not assigned (Moraxella)*
N.R.OTU7
230
0.43% Unassigned
Unassigned
not assigned (Neisseria)*
N.C.R.OTU1223
219
0.41% Unassigned
Unassigned
not assigned (Moraxella)*
N.C.R.OTU6242
218
0.40% Staphylococcaceae
Staphylococcus
Yes
#OTU ID
Seq.
DQ807326
14425
N.R.OTU4
Rel.
Abund.
Family (unless
otherwise specified)
Genus
153
10